Virulence gene regulation in Vibrio cholerae requires the action of four unusual transcription regulatory proteins, pairs of membrane localized transcription activator/effector molecules called ToxR/ToxS and TcpP/TcpH. Working models for the mechanisms of action of these proteins hold that ToxR and TcpP collaborate to activate expression of a protein ToxT, which activates genes encoding virulence factors cholera toxin and toxin co-regulated pilus. By contrast, ToxR alone - independently of TcpP - can regulate expression of OmpU and OmpT, two outer membrane proteins. Interactions between the activators and effectors are predicted to take place in the periplasmic space, although in general the roles of the effectors are less well characterized. TcpH acts to block a proteolytic mechanism that targets periplasmic domain of the TcpP. Whether TcpH plays any other role in the function of TcpP will be assessed in this study. FoxS may serve to confer higher order structure on ToxR essential for its function. Specific hypotheses generated from these mechanistic models of membrane-localized activator function will be tested. Much of what we understand about these proteins has come from genetic and biochemical studies, and these will continue in the work proposed herein. With advances and imaging and cytological resources available for studying the prokaryotic cell, studies aimed at developing reagents and experimental approaches for studying membrane localization of transcription complexes are also proposed. Specific Aims I. Determine the mechanism of ompU and toxT activation by ToxR. II. Determine the role of DNA binding and RNA polymerase interaction by TcpP for toxT activation. Ill. Define the mechanisms and consequences of activator/effector periplasmic interactions. IV. Develop cytological methods for analyzing membrane localized activator function.